The Introduction of Digital RNA Sequencing
RNA sequencing (RNA-seq) is the premier tool for mapping and quantifying transcriptomes by utilizing next-generation sequencing (NGS) technology. The transcriptome refers to the complete set of transcripts in a cell, which provides information on the transcript level for a specific developmental stage or physiological condition. Due to the bias of PCR amplification, different cDNA fragments are unevenly amplified. The easily amplified fragments are greatly enriched during the sequencing process, and some low-content fragments or fragments with severe base bias are even completely lost, which ultimately affects the accuracy of sequencing results. This only allows us to understand the general trend of gene expression, but it cannot satisfy the absolute quantification of the original gene expression level.
With UMI (Unique Molecular Identifier), we label every single molecule before library construction, such that each molecule consists of a unique sequence. Ideally, each template molecule can then be identified by its unique combination of the UMI sequence and the template sequence. After PCR amplification, PCR copies can be identified and eliminated from the dataset and hence uneven amplification and artifacts generated during the PCR can be almost completely eliminated. Quantitative statistics through UMI, the results are naturally more accurate. UMI combines NGS to study gene expression information of a certain species and tissue in a specific space-time state, and achieves high precision in sequence and quantification.
This is especially important for diagnostics as well as for imply of low amounts of starting material. UMI is highly recommended for the analysis of nucleotide populations containing numerous similar sequences, such as small-RNAs, ChIP-Seq tags, Aptamers, RAD-Seq tags or GBS-tags.
Key Features and Advantages
Digital RNA sequencing, also known as digital RNA-seq or UMI-RNA-Seq, is an absolute quantitative transcriptome sequencing technology, adding a unique molecular identifier (UMI) to each cDNA fragment before library amplification. UMI will accompany the entire process of fragment amplification, sequencing, and analysis. After sequencing, UMI is used to trace the source of each fragment, and combine fragments from the same source (with the same sequence and UMI) to accurately remove PCR amplification duplicates and accurately restore the original state of the sample before amplification. In this process, errors in PCR amplification and sequencing can also be corrected.
Figure 1. Transcriptome sequencing (left) and UMI-RNA-Seq (right)
CD Genomics provides full digital RNA sequencing service package including sample quality control, library construction, deep sequencing, raw data quality control, DEG analysis, and customized bioinformatics analysis. We can tailor this pipeline to your research interest. Please do not hesitate if you have any questions about our service.